Project Summary/Abstract. The broad, long-term goal of this project is to develop a comprehensive understanding of the developmental events that dictate cell fate decisions in formation of the synovial joint. Within this broad area, this proposal focuses on the differentiation of articular cartilage, which plays a central role in maintaining the low-friction environment of the joint space. Indeed, a hallmark of cells comprising the articular cartilage is their expression of proteoglycans, such as the protein lubricin, encoded by the Prg4 gene, that lubricates the joint and protects against the development of arthritis. Prg4 is specifically expressed in the superficial-most layer of the articular cartilage. Findings by both the Lassar lab and others have established that Prg4-expressing cells in the superficial zone of articular cartilage (in embryonic and early post-natal mice) serve as a stem cell population for all deeper regions of the articular cartilage in the adult. Thus, to elucidate how the articular cartilage stem cell population is both generated during development and maintained in the adult, a key objective of this proposal is to identify the factors that regulate the expression of both Prg4 and other genes that are specifically expressed in the superficial zone of the articular cartilage. Recent findings in the Lassar lab indicate that the transcription factor Creb5 is uniquely expressed in superficial zone articular chondrocytes (as opposed to both deeper zone articular chondrocytes and growth plate chondrocytes) and is a crucial regulator of Prg4 expression. Most notably, ectopic expression of Creb5 in deep zone bovine articular chondrocytes (which do not expression Prg4) enabled TGF-b2 and EGFR signals to induce Prg4 expression in these cells, to a level equal to that expressed by superficial zone articular chondrocytes. These findings suggest that Creb5 establishes a competent state in chondrocytes to express Prg4 in response to these signaling pathways. In addition, the Lassar lab has found that mice engineered to lack functional Creb5 fail to form many synovial joints, and that mis-expression of Creb5 throughout the limb bud mesenchyme (with Prx1-Cre) results in a profound loss of growth plate development in long bones. Taken together, these findings indicate that Creb5 plays a critical role in both the formation of synovial joints and is a both a novel and crucial regulator of Prg4/lubricin expression in articular chondrocytes. This project will identify both Creb5-dependent genes and the regulatory elements that drive the expression of these genes in primary bovine superficial zone articular chondrocytes; and mechanistically determine how TGFb, EGFR, and p38 signaling modulate the transcriptional activity of Creb5 in these cells. In addition, this project will determine how Creb5 regulates the formation of synovial joints, and elucidate how Creb5 expression in the epiphyseal perichondrium blocks extension of the growth plate into the epiphyseal ends of the long bones.